Dual role of melatonin as an anti-colitis and anti-extra intestinal alterations against acetic acid-induced colitis model in rats

The available ulcerative colitis drugs exhibit limited outcomes and adverse side effects. Therefore, our study aimed to investigate the therapeutic efficacy of melatonin in acetic acid (AA)-induced colitis to establish a possible treatment for colitis and its impacts on vital organs. Following colitis induction (2 ml 5% AA, rectally), rats were orally received melatonin (5 mg/kg) once per day for 6 days after colitis induction. Then, histopathological examination of colon, kidney, liver, and spleen was conducted, interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), and total antioxidant capacity (TAC) levels were assessed in colon tissue. Colitis induction in untreated rats caused necrotic effects in colon tissues, a significant increase in colonic IL-1β, TNF-α, MPO, and MDA levels, and a remarkable decrease in GSH and TAC levels in colon tissue in comparison to the control group. Meanwhile, melatonin treatment reversed these parameters by improving the microscopic and macroscopic colitis features and extra-intestinal (kidney, liver, and spleen) changes in all treated rats compared to the colitis control group. These results denote a reduction in colitis severity due to the anti-inflammatory and anti-oxidative effects of melatonin and its positive impact on the vital organs.

Animal handling and sample collection. Rats' weights (gram) were recorded daily throughout the experiment for observing weight change. Three hours after the last dose on day six, rats were euthanized by an overdose of sodium pentobarbital. Colons, spleens, kidneys, and livers of rats were rapidly excised and perfused in an ice-cold saline solution to remove red blood cells and clots. Proximal 8 cm of the colon was photographed to evaluate the macroscopic damage.
Assessment of pro-inflammatory and oxidative stress parameters. Half gram of each colon tissue was homogenized in 4.5 ml of 10 mmol cold Tris-HCl buffer (pH 7.4), then centrifugated at 3000 rpm at 4 °C for 10 min. The supernatant was transferred into aliquots and frozen at − 80 °C until used. Lowry's method 26 was performed for total protein content determination using a kit from Bio Basic Inc. (Cat. SK4031, Canada).

Myeloperoxidase (MPO), malondialdehyde (MDA), reduced glutathione (GSH), and total antioxidant capacity (TAC) levels.
Colonic MPO activity was determined using BioVision Inc. kit (Cat. E4581, CA, USA) according to the manufacturer's protocol. Colonic MDA was assessed spectrophotometrically using Ohkawa et al. method 27 using a kit from BioVision Inc. (Cat. K739, CA, USA). According to the manufacturer's protocol, the colonic GSH was measured using a kit from BioVision (Cat. K264, CA, USA), and TAC levels in colonic tissue were evaluated using a kit from LSBio Inc. (Cat. LS-F14457).
Pro-inflammatory cytokine assays. The colonic pro-inflammatory cytokine, interleukin-1beta, was measured by Rat IL-1β kit (Cat. ab100768, Abcam, USA), while tumor necrosis factor-alpha was assessed by rat TNF-α kit purchased from Bio-legend Inc. (Cat. 438,206, USA). Sandwich enzyme-linked immunosorbent assay (ELISA) was performed according to manufacture instructions. Absorbances were detected using an ELISA plate reader at a wavelength of 450 nm.
Histopathological examination. Organs of each rat (including Liver, Kidney, Spleen, and colon) were collected from all experimental groups at the end of the study, then fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned into 4-5 µm sections, deparaffinized in xylene, hydrated and stained with hematoxylin and eosin (H&E), and then examined by light microscope by a pathologist who is unware of the experimental design. Histopathological scoring of colitis was done according to Ahmed et al. 14  Statistical analysis. The IBM statistical package for the social sciences version 25 was used to analyze the data (copyright by IBM SPSS software, US). The Kolmogorov-Smirnov test was used to ensure that the data were normally distributed, then parametric statistical analysis was conducted. One-way analysis of variances (ANOVA) evaluated the changes between the experimental biomarkers. P < 0.05 was considered to be the minimal level of significance. A post hoc ANOVA (Tukey's homogeneity test) was employed to determine the differences and similarities between the experimental groups. The mean and standard error of the mean (SEM) were used to express all the results. GraphPad Prism version 5 was used to visualize the data (Graph Pad Software Inc., San Diego, CA). The improvement percent for each parameter was calculated according to the following equation: Ethics approval and consent to participate.

Results
Effect of melatonin on TAC and oxidative stress parameters (MPO, MDA, and GSH). When compared to the negative control group, the colitis-induced rats had a significant decrease (P < 0.0001) in TAC levels, a significant increase (P < 0.0001) in MPO and MDA levels, and a significant decrease (P < 0.001) in GSH levels. Meanwhile, when comparing the colitis + melatonin (5 mg/kg) group to the colitis group without treatment, a significant decline in MPO (P < 0.0001) (− 26.44%) and MDA (P < 0.001) (− 36.38%) levels, as well as a significant increase (P < 0.001) (+ 63.78%) in GSH levels, were noted. In addition, the treatment with melatonin resulted in a statistically significant increase (P < 0.0001) (+ 164.16%) in TAC levels compared to the colitisinduced group that was not treated. In contrast, no significant change was observed between the melatonintreated group after colitis induction with the negative and melatonin controls except for MPO and MDA, where a significant increase (P < 0.0001) was observed, as shown in Fig. 1A-D.

Effect of melatonin on pro-inflammatory cytokines (IL-1β and TNF-α). Acetic acid-induced coli-
tis in rats showed a significant increment (P < 0.0001) in IL-1β and TNF-α levels compared with the negative control group. Meanwhile, a significant decrease (P < 0.0001) in these parameters mentioned above was observed in the colitis + melatonin group (5 mg/kg, orally) (Improvement% for IL-1β -56.72%, for TNF-α -60.29%) compared with the colitis group without treatment. Besides, the treatment with melatonin had no significant change with the negative and melatonin control groups, as shown in Fig. 2A,B.
Histopathological features of colon and weight change. Upon microscopic examination (H&E X200 and X400), the colonic wall of the negative control and melatonin control groups revealed that the colonic wall had intact viable mucosa and superficial layer, normal muscularis mucosa, submucosa, and musculosa, with no inflammatory cellular infiltrate, and no inflammatory cellular infiltrate (Fig. 3A,B). In comparison to the negative control group, after 6 days of colitis induction with single instillation of acetic acid caused significant (P < 0.01) necrotic effects on the mucosa, submucosa, and musculosa, with a prominent inflammatory infiltration and dilated congested blood vessels (Fig. 3C). Meanwhile, when compared to the colitis control group, the melatonin-treated colitis group had a lower colitis parameter than the colitis control group (Fig. 3D). Bodyweight loss was reported following the instillation of acetic acid through the rectal route. The macroscopic features of the colons of the negative control and melatonin control groups were normal with no damage observed in the mucosa layer, while in the colitis control group, a severe edematous mucosal inflammation was observed; furthermore, this observation was reduced upon melatonin treatment in the melatonin treated group (Fig. 4A-D). The weights of the rats in both the negative control and the melatonin control groups increased over the treatment period while in the colitis intreated group, a weight loss was observed started from the second day till the end of the experiment, while this loss was inhibited in the melatonin treated group (Fig. 5A). The colitis treated with the melatonin group showed a lower score from the colitis control group -41.02% in terms of colitis severity, as presented in Fig. 5B.
Histopathological features of kidney, liver, and spleen. The glomeruli, tubules, and interstitium in kidney sections from the negative control and melatonin control groups (Fig. 6A,B) were of average size and shape. It was observed that the colitis-induced group had histopathological changes such as dilated congested blood vessels with hemorrhage and small-sized glomeruli with wide bowman's space (Fig. 6C). In contrast, no significant abnormalities were noticed when examining colitis rats administered with melatonin (Fig. 6D). It was shown that the central vein of the colitis-induced group was dilated and congested, with a hydropic alteration

Discussion
Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that generates inflammation and ulcers in the mucosa of the colon 1,2 . Because of the adverse side effects and restricted outcomes of the traditional treatments being used, it is necessary to study new product lines with more desirable therapeutic profiles to improve the outcomes 13 . According to the literature, several inflammatory responses in the inflamed mucosa have been shown to be modulated by ROS, stimulating the release of a wide range of inflammatory cytokines in a variety of tissues, hence exacerbating tissue damage. During oxidation, free radicals attack polyunsaturated fatty acids of the plasma membranes, causing their decomposition and deterioration. This mechanism is critical in the etiology of colitis 28 . Inflammatory markers in the colon involving MPO, an enzyme present in neutrophils, and MDA, one of the end products of lipid peroxidation; both have been employed as quantitative measures of colon inflammation 29,30 . GSH is a primary antioxidant agent that is crucial in the tissue repair process because it prevents free radicals from damaging the mucosa, which is essential for wound healing. During inflammation, GSH drops, leading to considerable colon mucosal deterioration. As a result, GSH is critical in protecting intestinal cells during instances of inflammation 31 . In our investigation, MPO and MDA levels were shown to be elevated in the colon tissue of the colitis control group, whereas GSH levels were found to be decreased. These observations come in parallel with prior investigations 14,32,33 . Moreover, the total antioxidant capacity (TAC) is reduced in both IBD phenotypes 7 . In our study, the TAC levels in the untreated colitis group were significantly lower compared to www.nature.com/scientificreports/ the negative control group, while a significant increment was observed in melatonin-treated rats. Furthermore, the observations of colon histology in the untreated colitis control group demonstrated an increase in inflammatory cells infiltration, leading to increased levels of oxidative stress. Also, ulcerative colitis is associated with extra-intestinal changes in vital organs such as the liver, spleen, and kidney; these alterations can be explained by the increased intestinal permeability and inflammatory reactions characteristic of ulcerative colitis. Moreover, our results explored that alterations in the histo-morphological structures of the liver, spleen, and kidney in the untreated colitis group, which is in agreement with previous studies [8][9][10][11]16 . Furthermore, the treatment with melatonin prevented these extra-intestinal alterations, as no histopathological changes were observed in these organs microscopically. Inflammatory reactions and cytokine profiles, such as IL-1β and TNF-α, are abundantly produced and associated with pathological alterations in IBD 34 . These cytokines drive downstream immune responses [35][36][37] . During active ulcerative colitis, IL-1β, one of the most prominent mediators of colon inflammation, is secreted in greater quantities 36 . Furthermore, macrophages and lymphocytes secrete TNF-α, an exceptionally strong cytokine, during the onset of an inflammatory response 38 . Additionally, TNF-α stimulates the production of pro-inflammatory cytokines, including IL-1β 39 . According to the current study, AA instillation in rats with colitis generated statistically significant rises in levels of IL-1β and TNF-α, which is similar to prior research by Ghasemi et al. 40 . Due to increased epithelial permeability, luminal antigens penetrate the lamina propria layer and activate the mucosal immune system, resulting in an over-reaction of the mucosal immune system, which results in the excessive production of pro-inflammatory cytokines [41][42][43][44][45] .
When the AA-induced colitis group was compared to the negative control group, the microscopic examination of the colon revealed significant necrosis in the mucosa layer with significant inflammatory cell infiltration in the AA-induced colitis group, which was not observed in the negative control group. According to the findings of Tahan et al. 46 , Rectal instillation with AA could cause severe mucosal injury, infiltration of pro-inflammatory cells, and ulceration. The increase in MPO, MDA, IL-1β, and TNF-α, as well as the decrease in GSH and TAC, were all observed in our study, supporting the microscopic examination.
Interestingly, the inhibition of IL-1β and TNF-α has been found to reduce colitis [47][48][49] . Remarkably, melatonin administration lowered IL-1β and TNF-α levels, which might be reasoned by the anti-inflammatory impact of melatonin on the inflamed colon. Our observation corresponds with a prior study 50 , where melatonin supplementation suppressed pro-inflammatory cytokines, including IL-1β and TNF-α, in the DSS-colitis model in rats. Also, treatment with melatonin induced a remarkable reduction in colitis histopathology's severity compared to the colitis control group. This impact is presumably attributable to the anti-inflammatory and antioxidant characteristics of melatonin [51][52][53] . Its impact on colitis was evidenced by improving the biochemical markers TAC and GSH and the decrease in IL-1β, TNF-α, MDA, and MPO compared to the colitis control group. In www.nature.com/scientificreports/ this context, neutrophil infiltration can be stimulated by TNF-α production 38 . In our investigation, neutrophil infiltration and TNF-α level decreased in the colon after treatments with melatonin. Also, the treatment with melatonin was found to augment and maintain the vital organs, including the liver, kidney, and spleen. Supporting that melatonin treatment has an anti-inflammatory and antioxidant effect on colons and the vital organs, as evidenced in the microscopic examination of these organs in the colitis treated with melatonin group compared to the untreated colitis group.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.